Generation of a novel functional neuronal circuit in Hoxa1 mutant

HAL Id: hal-00083754https://hal.archives-ouvertes.fr/hal-00083754

Submitted on 4 Jul 2006

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Generation of a novel functional neuronal circuit inHoxa1 mutant mice.

Eduardo Domínguez del Toro, Véronique Borday, Marc Davenne, RüdigerNeun, Filippo M. Rijli, Jean Champagnat

To cite this version:Eduardo Domínguez del Toro, Véronique Borday, Marc Davenne, Rüdiger Neun, Filippo M. Rijli, etal.. Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.. Journal of Neuroscience,Society for Neuroscience, 2001, 21 (15), pp.5637-5642. <hal-00083754>

https://hal.archives-ouvertes.fr/hal-00083754

https://hal.archives-ouvertes.fr

http://www.jneurosci.org/cgi/content/full/21/15/5637

Article paru dans le numéro 15 du volume 21 de The Journal of Neuroscience, année 2001 ; pp. 5637-5642 Copyright © 2001 Society for Neuroscience

1

Generation of a novel functional neuronal circuit

in Hoxa1 mutant mice Abbreviated title: Neuronal circuits function in Hoxa1 -/- mice 23 pages; abstract: 221words; introduction: 248 words; discussion: 431 words; 6 figures; no table. Correspondence: J. Champagnat, Institut de Neurobiologie Alfred Fessard, CNRS., UPR 2216 (bât. 33), 91198, Gif-sur-Yvette, France Phone: 33 (1) 69 82 34 06 Fax: 33 (1) 69 07 05 38 E-mail [email protected]

Eduardo Domínguez del Toro*1, Véronique Borday*1,3, Marc Davenne*2,4, Rüdiger Neun2, Filippo M. Rijli2, and Jean Champagnat1 1 Neurobiologie Génétique et Intégrative, UPR 2216 (bât. 33), C.N.R.S., 1, av. de la

Terrasse, 91198 Gif-sur-Yvette, France 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP,

Collège de France, BP 163-67404 Illkirch Cedex, CU de Strasbourg, France 3 Present address: Laboratoire de Biologie du Développement, Université Paris 7, case

7077, 2 place Jussieu, 75251, Paris Cedex 5, France. (V.B.). 4 Present address: Cold Spring Harbor Laboratory, 1, Bungtown Road, Cold Spring

Harbor, NY 11724, USA.(M.D.) * These first three authors contributed equally to this work ACKNOWLEDGEMENTS We wish to thank P. Chambon, G. Fortin, C. Goridis, R. Krumlauf, A. Lumsden for valuable discussions and comments on the manuscript. We also thank T. Jacquin for his participation in some in vitro experiments and M. Poulet for excellent technical assistance. We acknowledge the following colleagues for kind gifts of reagents: P. Chambon (Hoxa1 mice), R. Krumlauf (BGZ40 plasmid and Hoxb1 probe), J.F. Brunet (Phox2b probe). The 4D5 antibody was obtained from the Developmental Studies Hybridoma Bank under contract NO1-HD-7-3263. Work in J.C.’s laboratory was supported by a H.F.S.P. research grant 101/97, ACI BDPI #57, CNRS, Fondation pour la Recherche Médicale. E.D.T. was supported by CEE (BIO4-CT975-096) and FRM (EP001227/1) training grants. Work in F.M.R.’s laboratory was supported by CNRS, INSERM, Hôpital Universitaire de Strasbourg, Ligue Nationale Contre le Cancer (LNCC), Association pour la Recherche sur le Cancer, and Programme Génome du CNRS. M.D. was supported by fellowships from the LNCC and Fondation pour la Recherche Médicale (FRM). R.N. was supported by DAAD and FRM fellowships.

Keywords: Homeobox genes, Hoxa1 knockout; Respiration, Suction, Rhythm generation, Rhombomeres, Neural progenitors, Migratory pathways, Neuronal networks, Reticular formation, Pons, Hindbrain, Brainstem, Newborn mice.



2

ABSTRACT

The early organisation of the vertebrate brainstem is characterised by a cellular

segmentation into compartments, the rhombomeres, which follow a metameric

pattern of neuronal development. Expression of the homeobox genes of the Hox

family precedes rhombomere formation and analysis of mouse Hox mutations

revealed an important role in the establishment of rhombomere-specific neuronal

patterns. However, segmentation is a transient feature and a dramatic

reconfiguration of neurons and synapses takes place during fetal and postnatal

stages. Thus, it is not clear whether the early rhombomeric pattern of Hox

expression has any influence on the establishment of the neuronal circuitry of the

mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the

developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of

mouse Hoxa1-/- mutants has focused on early alterations of hindbrain

segmentation and patterning. Here, we show that ectopic neuronal groups in the

hindbrain of Hoxa1-/- mice establish a supernumerary neuronal circuit, that

escapes apoptosis and becomes functional postnatally. This system develops from

mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with

r2 identity, and integrates the rhythm-generating network controlling respiration

at birth. This is the first demonstration that changes in Hox expression patterns

allow the selection of novel neuronal circuits regulating vital adaptive behaviors.

The implications for the evolution of brainstem neural networks are discussed.



3

In the hindbrain of the vertebrate embryo, Hox genes are segmentally expressed and

loss- and gain-of-function mutations revealed their involvement in neuronal patterning

(e.g. Mark et al., 1993; Carpenter et al., 1993; Studer et al., 1996; Goddard et al., 1996;

Lumsden and Krumlauf, 1996; Gavalas et al., 1997, 1998; Rijli et al., 1998; Helmbacher

et al., 1998; Davenne et al., 1999; Rossel and Capecchi, 1999; Jungbluth et al., 1999;

Bell et al., 1999). Expression of Hoxa1 provides one of the earliest signs of

regionalisation within the developing hindbrain. As early as 7.5 day-post-coitum (dpc),

the Hoxa1 expression domain extends from the posterior end of the mouse embryo up to

the presumptive r3/r4 border and is downregulated before rhombomere boundary

formation (Murphy and Hill, 1991). This transient expression has a profound impact on

hindbrain patterning, as Hoxa1 targeted inactivation results in severe reduction of r4 and

r5 and their derived structures (e.g. the motor nucleus of the facial nerve) and in

lethality shortly after birth (Mark et al., 1993; Carpenter et al., 1993). However, it is

unclear how transient Hox expression before segment formation may influence the

generation of functional neuronal networks in the postsegmental hindbrain (Fortin et al.,

1999), and affect vital behaviors during postnatal life (Fortin et al., 2000). By

examining hindbrain neural networks in Hoxa1-/- mice, we now identify ectopic groups

of misspecified neurons which escape apoptosis (Rossel and Capecchi, 1999) during

development and control the respiratory rhythm-generating neural network

(Champagnat and Fortin, 1996) after birth.



4

MATERIALS AND METHODS

Mouse lines and genotyping.

Hoxa1 mutant mice (Mark et al., 1993), embryos, and newborns were PCR genotyped

as described (Gavalas et al., 1998). The r2-lacZ transgenic line was obtained by

injection of a construct carrying a 2.5 kb BamHI Hoxa2 genomic fragment (Frasch et

al., 1995) cloned in a non native orientation into the BGZ40 plasmid (Studer et al.,

1996), containing the human β-globin promoter driving lacZ expression. Transgenic r2-

lacZ mice were bred with Hoxa1+/- mice to produce Hoxa1+/-, r2-lacZ animals. The

latter were bred with Hoxa1+/- animals to produce embryos with the desired genotype.

Detection of the transgene was performed by PCR.

Whole-mount in situ RNA hybridisation, immunohistochemistry, and X-gal staining.

Whole-mount in situ RNA hybridisation was performed as described (Davenne et al.,

1999) using the Phox2b (Pattyn et al., 1997) and Hoxb1 (Studer et al., 1996) probes.

Whole-mount immunohistochemistry using the anti-ISL1 monoclonal antibody (4D5)

(Developmental Studies Hybridoma Bank) and X-gal staining were performed as

described (Davenne et al., 1999). Hindbrains were dissected out and flat-mounted

before being photographed. Postnatal neuronal groups (Jacquin et al., 1996) were

identified on coronal, horizontal and parasagittal 40 µm thick sections processed

alternatively using cresyl violet and polyclonal antibodies to choline acetyltransferase

(Chemicon International Incorporated, 1:1000 in PBS, pH 7.4) and to tyrosine

hydroxylase (Boehringer, 1:1000 in PBS), in the presence of Triton X-100 and



5

subsequently revealed using the Vectastain ABC kit (Vector) as described (Jacquin et

al., 1996). To study axonal pathways, the trigeminal motor root or the bulbar reticular

area ventral to the ambiguus nucleus was pressure injected with DiI (5mg/ml in DMSO)

after brain fixation. Incubation times (at 37°C) were 3 days after trigeminal injections

and 4 days after bulbar injections.

Plethysmograph recordings and naloxone treatment in vivo.

We have used 231 mice from 34 Hoxa-1 litters. Sixty mice were wild-type, 124

heterozygous and 47 homozygous mutants, a proportion close to the mendelian

expectation. Respiratory activity was measured every six hours using a modified

barometric method previously employed in neonates (Jacquin et al., 1996). The whole

body plethysmograph chamber (20 ml) equiped with a temperature sensor (LN 35 Z)

was connected to a reference chamber of the same volume. The pressure difference

between the two chambers was measured with a differential pressure transducer

(Validyne DP 103-12) connected to a sine wave carrier demodulator (Validyne, CD15).

Neonates were removed individually from the litter and placed in the plethysmograph

chamber kept hermetically closed and maintained at 31°C during the recording session

(2 min). During quiet breathing, a computer-assisted method was used to measure the

duration of inspirations and expirations from which the respiratory frequency is derived.

Naloxone was administered subcutaneously (3.33 mg/kg in 50 µl saline) using an

Hamilton syringe at the end of the first plethysmographic recording, 1-2 hours after

birth and the stimulatory effect on respiration was controlled 0.5-1 hour later.



6

Network analysis in vitro.

The brainstem was removed as previously (Jacquin et al., 1996, 1999) and cut

horizontally (Fig. 3f) under visual control with a vibratome (TPI, series 1000). The 1200

µm thick slice was transferred, dorsal side up, into a recording chamber and perfused

with an artificial cerebrospinal fluid (pH 7.4) containing (in mM) 130 NaCl, 5.4 KCl,

0.8 KH2PO4, 26 NaHCO3, 30 glucose, 1 MgCl2 and 0.8 CaCl2, saturated with carbogen

(90%02, 10% C02). Motor activities were recorded from the motor trigeminal roots

using suction electrodes. Previous experiments (Jacquin et al., 1999) have demonstrated

that the respiratory activity in vitro propagates to this nerve. The selected root was

contralateral to the studied Rpc-α/SNS (Fig. 3G), to avoid stimulating directly the

recorded motoneurons. Other electrodes were located on the dorsal surface under the

visual guidance of a microscope (ACM, Zeiss) and locations were identified

histologically. Neurons were recorded in the whole-cell configuration with patch-clamp

electrodes as in Fortin et al. (1999). Electrodes containing 0.1-0.5 mM AMPA in

artificial cerebrospinal fluid were used for pressure application (0.1 bar, 20 ms).

Experiments were performed according to autorisation by the ministry of Research-

Technology and Agriculture that provides autorisation to have the animal facilities.



7

RESULTS

A supernumerary neuronal structure in the dorsal Pons of Hoxa1 -/- mice

Morphological analysis of the Pons at birth indicates a rather extensive cellular

reorganisation in Hoxa1-/- mutants, affecting different cell types. First, in keeping with

the heterogeneous antero-posterior (A-P) pattern of the ventricular zone, the anterior

fourth ventricle exhibits a characteristic morphological abnormality in newborn mutants

(compare Fig. 1A,B,C,D). Moreover, the size of the reticular formation is affected both

dorsally and ventrally. Ventrally, a 40% reduction of the length of the ventral Pons (vP,

rectangle in Fig. 1E-F) results from the elimination of r4- and r5-derived structures. In

contrast, dorsally, a 6% increase of the postnatal A-P length of the Pons (dP, arrow in

Fig. 1E-F) was observed, so that the ratio dP/vP in Hoxa1-/-, although variable (average

± SEM: 1.45 ± 0.08, n=18), is much larger than in wild-type animals (0.77 ± 0.02,

n=18).

We have localised in the dorso-lateral Pons the anatomical modifications

underlying this dP increase. In wild-type mice, caudal to the trigeminal motor nucleus,

the Parvocellular Reticular Formation (Rpc-α, “pc” in Fig. 2A-B) normally contains

trigeminal pre-motor interneurons involved in feeding behaviors (Lund et al. 1998). The

Rpc-α is likely derived from r3, since it is eliminated in Krox-20-/- mutants (Jacquin et

al., 1996), in which pontine defects lead to an abnormal suction behavior after birth. In

all Hoxa1-/- mice (n=10), the anatomy of the Rpc-α is reorganised (Fig. 2) and extended

along the A-P axis, in keeping with the abnormalities of the r3-r4 region at early

developmental stages (described below, see also Mark et al., 1993; Carpenter et al.,



8

1993; Rijli et al., 1998; Gavalas et al., 1998; Helmbacher et al., 1998; Rossel and

Capecchi, 1999). In particular, radial stripes of reticular formation and ectopic

motoneurons alternate, forming a compound reticular and motor supernumerary

neuronal structure (SNS). Most extensive labelling of ectopic SNS motoneurons

included three distinct subnuclei (outlined in Fig. 2A-B) identified by analysis with anti-

cholineacetyltransferase antibodies. In addition, injecting the fluorescent marker DiI

into the trigeminal motor root (Fig. 2C-F) revealed that these ectopic subnuclei form a

distinct dorsoventral trigeminal motor fasciculus running laterally in the SNS (Fig. 2D

and F, stars) caudal to the normal root (Fig. 2E, star). Therefore, dP increase in Hoxa1-/-

mice results from the generation of three additional trigeminal subnuclei alternating

with stripes of reticular formation at the same location as the wild-type Rpcα.

Function of ectopic reticular neurons in the dorsolateral Pons of Hoxa1-/- mice

To further characterise the reticular cells of the SNS, we investigated their

functional connectivity (summarised in Fig. 3G). The hindbrain was isolated in vitro

during the first postnatal days (P0-P1) and the dorsal Pons was exposed in a thick

horizontal slice (Fig. 3F), and made accessible to dorsal approach under microscopic

control. This slice preparation also included the bilateral ventral respiratory group

(VRG, stars in Fig. 3G) that generates a persisting rhythmic activity propagating to

cranial (e.g. trigeminal) motor neurons from which it can be recorded (Jacquin et al.,

1996, 1999). Neuronal populations immediately caudal to the trigeminal nucleus (which

in wild-type include the Rpc-α premotor neurons; rectangle in Fig. 3G) were stimulated

by pressure application of the glutamatergic agonist α-amino-3-hydroxy-5-methyl-4-



9

isoxazoleproprionic acid (AMPA). The contralateral Vn was recorded to avoid direct

stimulation of motoneurons.

AMPA-induced non-rhythmic trigeminal activities recorded from the contralateral

trigeminal motor rootlet (the upward noisy deflection of the traces in Figs. 3A,C)

indicate that normal premotor Rpc-α inputs to the trigeminal motoneurons (Lund et al.,

1998) persist in Hoxa1-/- mutants. The Rpc-α normally lacks respiratory-related

functions. AMPA application had no effect on rhythm frequency in the wild-type

preparations (Fig. 3B). In the mutants, a robust increase in rhythm frequency is followed

in all cases by a transient inhibition of the rhythm (Fig. 3C, D). This effect strongly

suggests the presence of supernumerary functional efferent connections of the SNS to

the rhythm generator, resembling the wild-type ventral pontine respiratory connections,

located rostrally to the SNS and originating in r2 and r3 (Jacquin et al., 1996; Borday et

al., 1997). Moreover, rhythmic activity recorded from single neurons in the SNS area

(Fig. 3E) also indicated afferent connections from the rhythm generator. Furthermore,

abnormal axonal pathways were found in the lateral Pons by injecting the fluorescent

marker DiI into the VRG area (Fig. 3H-J). In the mutants, labelling from the VRG

revealed a robust axonal pathway (Fig. 3I), not present in the wild-type (Fig. 3H), and

running laterally in the Pons. Thus, in Hoxa1-/- mice the SNS exhibits a novel

relationship with the respiratory rhythm generator, while preserving premotor

connections with the trigeminal system.



10

Embryological origin of the supernumerary neuronal system

The appearance of this ectopic neuronal system prompts the question of its

embryological origin. We investigated the expression of rhombomere-specific

molecular markers in Hoxa1-/- mutant hindbrains (Fig. 4). Rhombomere-restricted gene

expression persists in the ventricular zone after the segmentation period (Wingate and

Lumsden, 1996). In 11.5 day post coitum (dpc) mutants, expression of the r4 marker

Hoxb1 is drastically reduced and patchy along the dorso-ventral axis (compare Fig. 4A-

B). To assay for r2 features, we generated a transgenic line containing the lacZ reporter

under the control of an Hoxa2 r2-specific enhancer (Frasch et al., 1995) (Fig. 4C). In

Hoxa1-/- mutants, ectopic patches of cells expressing the r2 marker are present at the r4

axial level (compare Fig. 4C-D), remarkably similar to what is observed in Hoxb1-/-

mice (Studer et al., 1996). In addition, patches of r2-like cells are also present at the

level of r3, as previously described (Helmbacher et al., 1998). Thus, in the absence of

Hoxa1, some neural precursors at the presumptive r3/r4 levels fail to activate or

properly maintain their appropriate molecular programs and acquire an r2 identity.

To investigate the developmental fate of these ectopic r2-like precursors we

examined motoneuron development in the hindbrain of Hoxa1-/- mice. In wild-type 11.5

dpc embryos, the Phox2b gene is expressed in migrating motoneurons (Pattyn et al.,

1997). Phox2b expression in ventral r4 identifies facial motoneurons migrating caudally

through r5 into r6 (bent arrow) to form the facial (VIIth) motor nucleus, whereas strings

of Phox2b-positive cells in r2 are indicative of dorsal migration of trigeminal

motoneurons (straight arrows, Fig. 5A). In the Hoxa1-/- mutant r4 region (Fig. 5B), a



11

much reduced, though not abolished, Phox2b expression identifies a small number of

facial motoneurons migrating caudally (bent and dashed arrow). In addition, an

abnormal trigeminal-like lateral migration of cells can be detected (straight arrows, Fig.

5B), which is completed at about 12.5 dpc (Fig. 5D) and results in a characteristic

dorso-lateral accumulation of ectopic Phox2b-positive cells (rectangle, compare Fig.

5C, D). This population includes ectopic motoneurons as assessed by anti-Islet1

immunohistochemistry (arrows; compare Fig. 5E, F). Remarkably, lack of caudal

migration of facial motoneurons and lateral trigeminal-like migration are also observed

in Hoxb1-/- mice (Studer et al., 1996). Thus, together with the above molecular analysis

(Fig. 4), these data suggest facial-to-trigeminal changes in motoneuron subtype identity

in Hoxa1 mutants which could be induced by lack of Hoxb1 activation in pre-r4 cells.

Persistence and functional role of the supernumerary neuronal system after birth

To investigate the functional role of the SNS in controlling respiratory and

feeding rhythms in vivo, we have compared mutant and wild type behaviors in relation

to the anatomical modification of the Pons. Although irregular after birth, the wild-type

minute ventilation increases progressively and stabilises at the end of the first day (Fig.

6A, left). In contrast, mutants exhibited a variable neonatal respiratory frequency

(NRF), 2-4 hours after birth, and eventually apneic breathing and death (Fig. 6A, right).

A correlation was found in mutants between the NRF and the hindbrain anatomical

index dP/vP (Fig. 6B, correlation coefficient: c=0.83 vs. c=0.32 in wild-type mice),

indicating that there are pontine abnormalities accelerating spontaneous breathing at



12

birth. In contrast, the suction behavior, estimated by the frequency of jaw openings

induced by a buccal stimulus (Jacquin et al., 1996), was normal in the mutants and

unrelated to dP/vP (c= 0.25). Furthermore, Hoxa1-/- newborns with a low NRF

(<35/min, n=7) died within 2.5 ± 0.8 hours (Fig. 6C, lower left triangles) while those

exhibiting a higher NRF (n=15) progressively increased their respiratory rate to normal

values (Fig. 6A, C) and survived for 18 ± 7 hours. Thus, one possibility is that the

appearance of the SNS may result in enhanced survival rates by significantly increasing

NRF values, so that the rhythm promoting action of the SNS seems to compensate lethal

apneic breathing resulting from vP hypoplasia. To further investigate this hypothesis,

animals with highest NRF were submitted to naloxone administration, a treatment

known to be effective on life-threatening pathologies resulting from the vP hypoplasia

as, for instance, in Krox-20-/- mutants (Jacquin et al., 1996). A striking effect of

naloxone administration was obtained in 2 out of the 5 treated Hoxa1-/- newborns (Fig.

6C, dots): one of them survived 4 days, whereas the other was sacrificed 12 days after

birth. Interestingly, in this animal, histological analysis revealed the same pattern of

SNS motoneurons (Fig. 6D) as observed at birth (Fig. 2). The survival of these

motoneurons is noteworthy, considering the wave of apoptosis which normally removes

abnormal motoneurons in the foetal hindbrain before birth (deLapeyrière and

Henderson, 1997). Altogether, the present in vitro and in vivo observations demonstrate

that the Hoxa1 mutation results in the incorporation of a SNS, which originates from the

mutant r3/r4 region, into the hindbrain neural network. As a consequence, the animal

acquires a novel respiratory-related function enhancing survival, while not affecting

suction, which is under the control of neuronal populations from the same region.



13

DISCUSSION

These results allow a hypothesis compatible with the involvement of

developmental control genes in the assembly of functional neuronal circuits (Tanabe

and Jessel, 1996; Brunet and Ghysen, 1999). In fact, this work provides the first formal

evidence that the selective modification of the expression pattern of a Hox gene whose

expression is transient in the presumptive hindbrain, namely Hoxa1, is sufficient to

incorporate a novel functional neural circuit in the mature hindbrain. This striking

finding prompts the question of the cascade of regulatory events triggered by Hoxa1

loss-of-function, leading to such long-term modification of hindbrain neural networks.

Previous work demonstrated a role for Hoxa1 in the activation of Hoxb1 expression in

the presumptive r4 (Studer et al., 1998). Thus, some of the long-term effects of the

Hoxa1 mutation could be due to the lack of Hoxb1 activation in a subset of presumptive

r4 cells, leading to r2-like specification. However, Hoxa1, unlike Hoxb1, appears to

control both r4 and, indirectly, r3 development (Helmbacher et al., 1998; this study).

Thus, it is tempting to speculate that regulatory changes in two adjacent rhombomeres

may be required for the generation of a SNS. Interestingly, we have recently shown that

assembling of a rhythm-promoting respiratory network also requires a two-segment

functional unit in the chick (Fortin et al., 1999). In this respect, it will be interesting to

compare the physiology of neuronal networks in Hoxb1-/- mutants to that of Hoxa1-/-

mutants.



14

Because hindbrain neurons control adaptive behaviors, these findings have

considerable significance both on developmental and evolutionary grounds. The

evolution of neural networks of multisegmental origin may be facilitated by the

partitioning of the early hindbrain in a number of metameric units initially developing

as independent modules (Lumsden, 1990; Clarke and Lumsden, 1993; Champagnat and

Fortin, 1996). As a result, subsets of neurons may be developmentally isolated from

each other and allowed to evolve independently. Our present data suggest that Hox

genes may provide a genetic basis for segment-specific modulation of neuronal

development and connectivity. Changes in Hox cis-regulatory modules and downstream

targets have been suggested to underlie morphological changes of segmented structures

in animal evolution (Gellon and McGinnis, 1998). Similarly, local changes in the

regulation of Hox genes within the segmented hindbrain of vertebrates may open novel

opportunities for the evolution of distinct subsets of neurons, without affecting the

function of others, eventually resulting in novel functional features (see Brunet and

Ghysen, 1999). In this respect, studies of conditional segment-specific Hox mutations,

which may not result in lethality of the animal, will be important to further investigate

adaptive mechanisms in development of hindbrain neuronal networks.


Clarke JD, Lumsden A (1993) Segmental repetition of neuronal phenotype sets in the

chick embryo hindbrain. Development 118:151-162.


15

REFERENCES

Bell E, Wingate RJ, Lumsden A (1999) Homeotic transformation of rhombomere

identity after localized Hoxb1 misexpression. Science 284:2168-71.

Borday V, Kato F, Champagnat J (1997) A ventral pontine pathway promotes rhythmic

activity in the medulla of neonate mice. NeuroReport 8 : 3679-3683.

Brunet JF, Ghysen A (1999) Deconstructing cell determination: proneural genes and

neuronal identity. Bioessays 21:313-8.

Carpenter EM, Goddard JM, Chisaka O, Manley NR, Capecchi MR (1993) Loss of

HoxA1 (Hox-1.6) function results in the reorganization of the murine hindbrain.

Development 118:1063-1075.

Champagnat J, Fortin G (1996) Primordial respiratory-like rhythm generation in the

vertebrate embryo. Trends in Neurosciences 20:119-124.

Davenne M, Maconochie MK, Neun R, Pattyn A, Chambon P, Krumlauf R, Rijli FM

(1999) Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the

rostral hindbrain. Neuron 22:677-91.

De Lapeyrière O, Henderson CE (1997) Motoneuron differentiation, survival and

synaptogenesis. Curr. Opinion Genet. Dev. 7:642-650.



16

Fortin G, Jungbluth S, Lumsden A, Champagnat J. (1999) Segmental specification of

GABAergic inhibition during development of hindbrain neural networks. Nature

Neuroscience 2:873-877.

Fortin G, Domínguez del Toro E, Abadie V, Guimarães L, Foutz AS, Denavit-Saubié

M, Rouyer F, Champagnat J (2000) Genetic and developmental models for the neural

control of breathing in vertebrates. Respiration Physiology 122:247-257.

Frasch M, Chen X, Lufkin T (1995) Evolutionary-conserved enhancers direct region-

specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila.


Gavalas A, Davenne M, Lumsden A, Chambon P, Rijli FM (1997) Role of Hoxa-2 in

axon pathfinding and rostral hindbrain patterning. Development 124:3683-3691.

Gavalas A, Studer M, Lumsden A, Rijli FM, Krumlauf R, Chambon P (1998) Hoxa1

and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal

arch. Development 125:1123-1136.

Gellon G, McGinnis W (1998) Shaping animal body plans in development and

evolution by modulation of Hox expression patterns. Bioessays 20:116-25.

Goddard JM, Rossel M, Manley NR, Capecchi MR (1996) Mice with targeted

disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve. Development

122:3217-28.



17

Helmbacher F, Pujades C, Desmarquet C, Frain M, Rijli FM, Chambon P, Charnay P

(1998) Hoxa1 and Krox-20 synergize to control the development of rhombomere 3.


Jacquin TD, Borday V, Schneider-Maunoury S, Topilko P, Ghilini G, Kato F, Charnay

P, Champagnat J (1996) Reorganisation of pontine rhythmogenic neuronal networks in

Krox-20 knockout mice. Neuron 17:747-758.

Jacquin TD, Sadoc G, Borday V, Champagnat J (1999) Pontine and medullary control

of the respiratory activity in the trigeminal and facial nerves of the newborn mouse: an

in vitro study. European Journal of Neuroscience 11:213-222.

Jungbluth S, Bell E, Lumsden A (1999) Specification of distinct motor neuron identities

by the singular activities of individual Hox genes. Development 126:2751-8.

Lumsden A (1990) The cellular basis of segmentation in the developing hindbrain.

Trends in Neurosciences 13:329-35.

Lumsden A, Krumlauf R (1996) Patterning the vertebrate neuraxis. Science 274:1109-

1115.

Lund JP, Kolta A, Westberg KG, Scott G (1998) Brainstem mechanisms underlying

feeding behaviors, Current Opinion in Neurobiology 8:718-724.

Mark M, Lufkin T, Vonesh JL, Ruberte E, Olivo JC, Dollé P, Gorry P, Lumsden A,

Chambon P (1993) Two rhombomeres are altered in Hoxa-1 mutant mice. Development

119:319-338.



18

Murphy P, Hill RE (1991) Expression of the mouse labial-like homeobox-containing

genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain. Development

111:61-74.

Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF (1997) Expression and interactions

of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis.


Rijli FM, Gavalas A, Chambon P (1998). Segmentation and specification in the

branchial region of the head: the role of the Hox selector genes. Int. J. Dev. Biol.

42:393-401.

Rossel M, Capecchi MR (1999) Mice mutant for both Hoxa1 and Hoxb1 show

extensive remodeling of the hindbrain and defects in craniofacial development.


Studer M, Gavalas A, Marshall H, Ariza-McNaughton L, Rijli FM, Chambon P,

Krumlauf R (1998) Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in

regulation of early hindbrain patterning. Development 125:1025-1036.

Studer M, Lumsden A, Ariza-McNaughton L, Bradley A, Krumlauf R (1996) Altered

segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1.

Nature 384:630-634.

Tanabe Y, Jessell TM (1996) Diversity and pattern in the developing spinal cord.

Science 274:1115-1123.



19

Wingate RJT, Lumsden A (1996) Persistence of rhombomeric organisation in the

postsegmental hindbrain. Development 122:2143-2152.



20

FIGURE LEGENDS

Fig. 1. Distinct dorsal and ventral anatomical phenotypes in the Hoxa1-/- brainstem at

birth. A-D: adjacent horizontal sections (A and C are dorsal to B and D, respectively;

scale bar: 250µm) showing the ependymal epithelium at the anterior end of the fourth

ventricle (see also E, F caudal to DTg and Fig. 2B for location): it forms, in WT mice

(A, B, E), a single invagination closing in the dorsal Pons medially to the trigeminal

motor nucleus and multiple invaginations (2 to 5) in Hoxa1-/- mice (C, D, F). E, F:

Parasagittal sections of the hindbrain at P0 in WT (E) and Hoxa1-/- (F) littermates; A-P

length of the Pons is differently affected dorsally (arrow, from the rostral limit of the

hypoglossal nucleus, 12, to the caudal limit of the dorsal tegmental area, DTg) and

ventrally (rectangle, from the rostral pole of the inferior olive, IO, to the caudal pole of

the pontine nuclei, Pn); IP: interpeduncular nucleus, Sol: solitary nucleus.

Fig. 2. The dorsal anatomical phenotype in Hoxa1-/- mice at birth: identification of

motoneurons showing location of the supernumerary neuronal structure (SNS). A:

sagittal sections of the brainstem, cut parallel to Fig. 1, E and F. The drawings on the

left (including the analysis of 5 Hoxa1-/- mice) include both lateral and medial structures

(scale bar, 1 mm). Medial sections (in gray, showing the ventricular surface) are

illustrated in Fig. 1, E and F: note that supernumerary motor (lateral) and ventricular

(medial) structures are at the same antero-posterior level of the dorsal Pons. Lateral

sections (on the right) show cholineacetyltransferase immunoreactive WT (+/+) ventral

facial structures eliminated by the mutation: the branchial motor nucleus (VII), the



21

preganglionic nucleus (pg) and accessory nuclei (between VII and pg, extending close

to the descending facial root, VIIn). In Hoxa1-/- (-/-), caudal to the trigeminal nucleus

(V), the SNS includes three dorsal motor subnuclei (outlined and numbered) alternating

with 2 unstained stripes of reticular formation. Abbreviations IP, Pn, IO: as in Fig. 1;

X, XII: dorsal vagal and hypoglossal motor nuclei; SO: superior olive; pc: parvocellular

reticular formation. B: horizontal sections cut parallel to the arrow in Fig. 1, E and F.

Drawings on the left (including the analysis of 5 Hoxa1-/- mice) show the left part of the

Pons (scale bar: 1 mm) and the relative positions of the V and VII nuclei and trigeminal

nerve root (Vn). Note, close to the midline (dotted line), appearance of a supernumerary

ventricular structure (illustrated in Fig. 1D) and elimination of the abducens motor

nucleus (VI). The right part superimposes cholineacetyltransferase immunoreactive

pontine neurons in WT (black) and Hoxa1-/- (red) littermates, from 4 horizontal sections

sampling, in each littermate, the entire V nucleus and adjacent areas. Supernumerary

motor nuclei n°1, 2 and 3 are at the same place as the WT Rpc-α (pc), VIIn and pg,

respectively. C-E: horizontal sections showing retrograde DiI labelling of trigeminal

and SNS motoneurons in a WT (C, arrow: 200µm) and an Hoxa1-/- (D, E) mouse.

Labelling of the SNS shows the three ectopic trigeminal subnuclei (compare D to C)

and a more ventral view (E) shows a supernumerary dorso-ventral fasciculus located

laterally in subnucleus n°2 (star in D and E) and distinct from the WT-like Vn. F:

medial half of a subnucleus n°2 at higher magnification (arrow: 67 µm, oriented as in C;

the border of the V is in the upper left corner; subnucleus n°1 is lacking). The

supernumerary motoneuron (triangle) shows an axon (stars) running in the direction of

the lateral fasciculus.



22

Fig. 3. Functional connectivity of reticular neurons in the Hoxa1-/- supernumerary

neuronal structure at birth. A-D: modification of the contralateral trigeminal nerve

activity (Vn) induced by excitating SNS neuronal cell bodies using brief (25 ms)

pressure applications of AMPA in WT (A, B) and Hoxa1-/- (C, D) hindbrain slices in

vitro. A, C: 4 samples of integrated Vn activity (2 min long) starting (from top to

bottom) -2, 0, 3 and 5 min after AMPA application (time indicated on the left). In both

WT and Hoxa1-/-, the rhythm generator produces bursts of activity (fast upward

deviations) and AMPA generates background non-rhythmic activity starting at time 0

min. B, D: temporal evolution (scale bar: 2 min) of average (± standard error) burst

frequency from 5 experiments. Significant increase followed by inhibition (p<.001)

indicates a functional connection to the rhythm generator in Hoxa1-/- but not in WT

mice. E: Vn: integrated nerve activity; Em: membrane potential of a single (Hoxa1-/-)

neuron located in the SNS area (scale bars: 20mV, 1s): a connection from the rhythm

generator results in a simultaneous Vn burst and neuronal depolarization inducing firing

of action potentials. F, G: schematic presentation of the slice preparation in sagittal (F,

arrowhead indicates the upper side) and horizontal (G) sections. Rectangle in G:

approximate extent of the area affected by AMPA applications (arrowhead, more medial

applications were ineffective); thin arrows: WT projections, preserved in mutants: these

are either rhythmic, from the bilateral rhythm generator (stars) to the contralateral

trigeminal nucleus (VMo) and Vn (recorded) or non-rhythmic premotor from Rpc-

α/SNS to VMo; thick arrows: supernumerary connections in mutants including those

from SNS to the rhythm generator and the trigeminal axons of SNS motoneurons. H-J:

Sagittal sections (location in J; rostral to the left) of the most lateral 300µm of the Pons



23

showing in mutant (I), but not in WT (H) animals, an axonal fasciculus stained after DiI

injection in the area of the rhythm generator (lower right corner); scale bar: 200µm.

Fig. 4. Molecular and morphological patterning defects in Hoxa1 mutant hindbrain.

Dorsal view of 11.5 d.p.c. wild-type (WT, A, C) and Hoxa1-/- (B, D) mutant hindbrains

hybridized with the r4-specific Hoxb1 (A, B) or carrying a lacZ reporter under the

control of an r2-specific enhancer (C, D). Vertical arrows indicate location of the

motoneuron progenitor columns.

Fig. 5. The Hoxa1-/- supernumerary motoneurons: migration and final postnatal

location. Dorsal view of 11.5 (A, B) and 12.5 (C-F) d.p.c. WT and Hoxa1-/- mutant

hindbrains, respectively, flat-mounted and hybridized with Phox2b (A-D) or Isl1 (E, F)

probes; bent white arrows in A, B: caudal migration of facial (VII) motoneurons;

straight arrows in A, B: dorsal migration of trigeminal (V) motoneurons and, in Hoxa1-/-

(B), of supernumerary motoneurons from r4; rectangle in C, D and arrows in E, F:

ectopic, dorso-lateral, accumulation of Phox2b and Isl1 positive cells, not present in

WT.

Fig. 6. The Hoxa1-/- breathing pattern after birth. A: samples of plethysmographic

recording (inspiration upwards) 2, 6, 12, 18 and 24 hours after birth (p.n.) showing

normal maturation in a WT and transient increase of frequency in a mutant (scale bars

20 µl, 1s). The mutant typically exhibits irregular breathing at birth (top trace), and

eventually (bottom trace) apneic breathing and death. B: Individual Hoxa1-/- (empty

triangles) and WT (black squares) mice identified by their respiratory rate at birth

(ordinates) and the dP/vP index quantifying abnormality of the pontine A-P distances



24

(see arrow (dP) and rectangle (vP) in Fig. 1E,F): a correlation exists in Hoxa1-/-, not in

WT mice. C: Temporal evolution of average respiratory frequency (± SEM) in Hoxa1-/-

animals breathing faster or slower than 35/min at birth (empty triangles): slowest

animals lack rhythm stimulation shown in A (6-18); fastest animals survive longer;

death has been delayed by > 3 d.p.n. in 2 animals (dots) treated with subcutaneous

naloxone (NLX). D: Supernumerary motoneurons in a NLX-treated animal sacrificed

12 days after birth: sagittal section (rostral to the left) showing cholineacetyltransferase

immunoreactive motoneurons (arrowheads), caudal to the trigeminal motor nucleus

(located in the upper left corner, scale bar: 100µm).

Documents

Generation of a novel functional neuronal circuit in Hoxa1 mutant